综述与专题评论

产环糊精葡萄糖基转移酶的菌株选育及酶分子改造研究进展

  • 陶大炜 ,
  • 张颖琦 ,
  • 赵赛赛
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  • 1(上海市虹口区疾病预防控制中心,上海,200082)
    2(上海海洋大学 食品学院,上海,201306)
第一作者:硕士,技师(通信作者,E-mail:tdw051012@163.com)

收稿日期: 2023-10-06

  修回日期: 2023-10-23

  网络出版日期: 2024-08-21

Research progress in breeding of cyclodextrin glycosyltransferase producing strains and enzyme molecular modification

  • TAO Dawei ,
  • ZHANG Yingqi ,
  • ZHAO Saisai
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  • 1(Shanghai Hongkou District Center for Disease Control and Prevention, Shanghai 200082, China)
    2(College of Food Sciences and Technology, Shanghai Ocean University, Shanghai 201306, China)

Received date: 2023-10-06

  Revised date: 2023-10-23

  Online published: 2024-08-21

摘要

环糊精(cyclodextrin,CD)因其内疏水外亲水的特殊结构被广泛运用,而酶法制备CD是常用方法,主要应用环糊精葡萄糖基转移酶(cyclodextrin glycosyltransferase,CGT酶)。该文对产CGT酶的菌株选育和酶分子改造这两方面进行综述,重点讨论提升酶表达量的4种异源表达常用优化策略:产酶条件优化、信号肽选择、启动子优化、密码子优化,以及定点突变、定点饱和突变、易错PCR这3种常用的酶分子改造方法对酶相关特性的影响。

本文引用格式

陶大炜 , 张颖琦 , 赵赛赛 . 产环糊精葡萄糖基转移酶的菌株选育及酶分子改造研究进展[J]. 食品与发酵工业, 2024 , 50(15) : 359 -371 . DOI: 10.13995/j.cnki.11-1802/ts.037550

Abstract

Cyclodextrin (CD) is widely used due to its unique structure of internal hydrophobicity and external hydrophilicity, and enzymatic preparation of CD is a commonly used method, mainly uses cyclodextrin glycosyltransferase (CGTase).This paper reviews the breeding of CGTase producing strains and enzyme molecular modification, focusing on the discussion of four commonly used optimization strategies for heterologous expression to increase enzyme expression:enzyme production conditions optimization, signal peptide selection, promoter optimization, codon optimization, as well as the impact of three commonly used enzyme molecular modification methods on enzyme related characteristics:site-directed mutation, site-saturation mutation, and error prone PCR.

参考文献

[1] 李兆丰. 软化类芽孢杆菌α-环糊精葡萄糖基转移酶在大肠杆菌中的表达及其产物特异性分析[D].无锡:江南大学, 2009.
LI Z F.Expression of α-cyclodextrin glycosyltransferase from Paenibacillus macerans in Escherichia coli and analysis of its product specificity[D].Wuxi:Jiangnan University, 2009.
[2] 李才明. β-环糊精葡萄糖基转移酶在枯草杆菌中的分泌表达及其热稳定性研究[D].无锡:江南大学, 2014.
LI C M.Secretory expression in Bacillus subtilis and thermostability of β-cyclodextrin glycosyltransferase[D].Wuxi:Jiangnan University, 2014.
[3] 王蕾. 环糊精葡萄糖基转移酶的产物特异性分子改造及发酵制备研究[D].无锡:江南大学, 2018.
WANG L.Product specificity engineering and fermentation of cyclodextrin glycosyltransferase[D].Wuxi:Jiangnan University, 2018.
[4] 陈龙然. 产α-环糊精葡萄糖基转移酶的菌株分离、产酶条件优化与酶学特性研究[D].杭州:浙江大学, 2005.
CHEN L R.Breeding and optimized fermentation of an α-glucanotransferase producing strain and its enzymatic characteristics[D].Hangzhou:Zhejiang University, 2005.
[5] 金征宇, 柏玉香, 王金鹏.环糊精系列综述之一 环糊精葡萄糖基转移酶的筛选及其定向改造[J].食品与生物技术学报, 2012, 31(2):113-123.
JIN Z Y, BAI Y X, WANG J P.Screen and modification of cyclodextrin glycosyltransferase[J].Journal of Food Science and Biotechnology, 2012, 31(2):113-123.
[6] TILDEN E B, HUDSON C S.Preparation and properties of the amylases produced by Bacillus macerans and Bacillus polymyxa[J].Journal of Bacteriology, 1942, 43(4):527-544.
[7] 朱国威, 李丽芬, 傅楠楠, 等.产环糊精葡萄糖基转移酶苦橙内生细菌的分离鉴定[J].食品研究与开发, 2023, 44(14):198-204.
ZHU G W, LI L F, FU N N, et al.Isolation and identification of endophytic bacteria producing cyclodextrin glucosyltransferase from bitter orange[J].Food Research and Development, 2023, 44(14):198-204.
[8] 张新武. 常压室温等离子诱变选育β-环糊精葡萄糖基转移酶高产菌株及发酵工艺研究[J].食品安全质量检测学报, 2016, 7(12):4930-4938.
ZHANG X W.Study on high-yield strains producing β-cyclodextrin glycosyltransferase by atmospheric room temperature plasma mutation breeding and optimization of their fermentation conditions[J].Journal of Food Safety & Quality, 2016, 7(12):4930-4938.
[9] 张洪斌, 凌国庆, 胡雪芹, 等.Ñ+注入诱变选育β-环状糊精葡萄糖基转移酶高产菌株及其发酵条件优化[J].食品科学, 2012, 33(15):239-245.
ZHANG H B, LING G Q, HU X Q, et al.Screening of a high-yield β-cyclodextrin glycosyltransferase-producing Bacillus cereus strain and optimization of its fermentation conditions[J].Food Science, 2012, 33(15):239-245.
[10] 陶大炜, 张小丹, 宁喜斌, 等.复合诱变选育高产α-环糊精葡萄糖基转移酶的菌株及产酶条件优化[J].食品与发酵工业, 2021, 47(19):63-70.
TAO D W, ZHANG X D, NING X B, et al.Strain breeding for high-yielding α-cyclodextrin glucosyltransferase and optimization of the enzyme formation conditions[J].Food and Fermentation Industries, 2021, 47(19):63-70.
[11] 张智维, 雷新辉, 张海群.紫外线和亚硝酸诱变选育高产α-环糊精葡萄糖基转移酶菌株[J].粮油食品科技, 2013, 21(5):101-104.
ZHANG Z W, LEI X H, ZHANG H Q.Breeding of high yield α-cyclodextrin glucosyltransferase strains by UV and nitrite mutagenesis[J].Science and Technology of Cereals, Oils and Foods, 2013, 21(5):101-104.
[12] 廖威, 华慧颖, 莫于旺.一株β-环状糊精葡萄糖基转移酶产生菌的筛选与发酵条件研究[J].轻工科技, 2011,27(002):1-3.
LIAO W, HUA H Y, MO Y W.Screening and fermentation conditions of a β-cyclodextrin glucosyltransferase producing strain[J].Guang Xi Journal of Light Industry, 2011, 27(002):1-3.
[13] 张晓磊. 一株α-环糊精葡萄糖基转移酶产生菌的选育,鉴定,产酶条件优化和酶学性质的研究[D].上海:上海海洋大学, 2015.
ZHANG X L.Breeding, identification and optimized fermentation of an alpha cyclomaltodextrin glucanotransferase producing strain and its enzymatic characterization[D].Shanghai:Shanghai Ocean University, 2015.
[14] 范博望. Bacillus stearothermophilus环糊精葡萄糖基转移酶在短小芽孢杆菌中的重组表达、发酵优化及应用研究[D].无锡:江南大学, 2016.
FAN B W.Fermentation and application of CGTase from Bacillus stearothermophilus expressed in Bacillus brevis[D].Wuxi:Jiangnan University, 2016.
[15] 刘花. 基于密码子优化的α-环糊精糖基转移酶在大肠杆菌及巨大杆菌中的表达[D].无锡:江南大学, 2012.
LIU H.Codon usage optimization aided α-cyclodextrin glycosyltransferase expression in E.coli and B.megaterium[D].Wuxi:Jiangnan University, 2012.
[16] WANG L, CHEN S, WU J.Cyclodextrin enhanced the soluble expression of Bacillus clarkii γ-CGTase in Escherichia coli[J].BMC Biotechnology, 2018, 18(1):72.
[17] 周媛媛. 来源于Bacillus xiaoxiensis STB08的β-CGT酶在大肠杆菌中的分泌表达及发酵优化[D].无锡:江南大学, 2022.
ZHOU Y Y.Secretion expression in Escherichia coli and fermentation optimization of β-CGTase from Bacillus xiaoxiensis STB08[D].Wuxi:Jiangnan University, 2022.
[18] 李晓涵, 郝建华, 郭姣梅, 等.环糊精葡萄糖基转移酶高效异源表达研究进展[J].微生物学通报, 2020, 47(2):615-622.
LI X H, HAO J H, GUO J M, et al.Advance in high-level heterologous expression of cyclodextrin glycosyltransferase[J].Microbiology China, 2020, 47(2):615-622.
[19] SONG K, SUN J J, WANG W, et al.Heterologous expression of cyclodextrin glycosyltransferase my20 in Escherichia coli and its application in 2-O-α-D-glucopyranosyl-L-ascorbic acid production[J].Frontiers in Microbiology, 2021, 12:664339.
[20] ZHANG J G, ZHAO Y X, LI M L, et al.Optimization of defined medium for recombinant Komagataella phaffii expressing cyclodextrin glycosyltransferase[J].Biotechnology Progress, 2019, 35(5):e2867.
[21] GIMENEZ G G, COSTA H, DE LIMA NETO Q A, et al.Sequencing, cloning, and heterologous expression of cyclomaltodextrin glucanotransferase of Bacillus firmus strain 37 in Bacillus subtilis WB800[J].Bioprocess and Biosystems Engineering, 2019, 42(4):621-629.
[22] 陈龙军, 林陈强, 张慧, 等.嗜热芽胞杆菌α-环糊精葡萄糖基转移酶在枯草芽胞杆菌中的表达[J].福建农业学报, 2019, 34(5):600-605.
CHEN L J, LIN C Q, ZHANG H, et al.Expression of α-cyclodextrin glycosyltransferase gene of Gebacillius sp.CHB1 in Bacillus subtilis[J].Fujian Journal of Agricultural Sciences, 2019, 34(5):600-605.
[23] 柴宝成. 分子改造环糊精葡萄糖基转移酶合成糖基化染料木素[D].无锡:江南大学, 2021.
CHAI B C.Engineering of cyclodextrin glucosyltransferase for the synthesis of glycosylated genistein[D].Wuxi:Jiangnan University, 2021.
[24] 郑金珠, 吴华伟, 李相前.培养基添加剂促进重组大肠杆菌胞外分泌β-环糊精葡萄糖基转移酶的工艺优化[J].食品科技, 2019, 44(11):16-22.
ZHENG J Z, WU H W, LI X Q.Optimization of medium additives for promoting extracellular secretion of β-cyclodextrin glucosyltransferase from recombinant Escherichia coli[J].Food Science and Technology, 2019, 44(11):16-22.
[25] ZHENG J Z, LI X Q, WU H W.High-level extracellular secretion and characterization of the thermophilic β-cyclodextrin glucanotranferase from Paenibacillus campinasensis in Escherichia coli[J].3 Biotech, 2019, 9(10):372.
[26] MAHMUD H, ISMAIL A, ABDUL RAHIM R, et al.Enhanced secretion of cyclodextrin glucanotransferase (CGTase) by Lactococcus lactis using heterologous signal peptides and optimization of cultivation conditions[J].Journal of Biotechnology, 2019, 296:22-31.
[27] SONNENDECKER C, WEI R, KURZE E, et al.Efficient extracellular recombinant production and purification of a Bacillus cyclodextrin glucanotransferase in Escherichia coli[J].Microbial Cell Factories, 2017, 16(1):87.
[28] 陈龙军, 林陈强, 贾宪波, 等.信号肽筛选优化提高耐热α-环糊精酶在枯草芽胞杆菌中的表达[J].福建农业学报, 2022, 37(3):414-422.
CHEN L J, LIN C Q, JIA X B, et al.Enhanced thermophilic α-cyclodextrin glycosyltransferase expression by optimizing target signal peptide in Bacillus subtilis[J].Fujian Journal of Agricultural Sciences, 2022, 37(3):414-422.
[29] PETROVA P, TONKOVA A, PETROV K.Sequence analysis, cloning and extracellular expression of cyclodextrin glucanotransferase gene from the alkaliphilic Bacillus pseudalcaliphilus 8SB in Escherichia coli[J].Process Biochemistry, 2012, 47(12):2139-2145.
[30] 李云菲, 宿玲恰, 吴敬.启动子对枯草芽孢杆菌表达环糊精葡萄糖基转移酶的影响[J].基因组学与应用生物学, 2019, 38(8):3540-3547.
LI Y F, SU L Q, WU J.Effect of promoter on the expression of cyclodextrin glucosyltransferase by Bacillus subtilis[J].Genomics and Applied Biology, 2019, 38(8):3540-3547.
[31] ZHANG K, SU L Q, DUAN X G, et al.High-level extracellular protein production in Bacillus subtilis using an optimized dual-promoter expression system[J].Microbial Cell Factories, 2017, 16(1):32.
[32] DENG C, LI J H, SHIN H D, et al.Efficient expression of cyclodextrin glycosyltransferase from Geobacillus stearothermophilus in Escherichia coli by promoter engineering and downstream box evolution[J].Journal of Biotechnology, 2018, 266:77-83.
[33] ZHANG J G, ZHANG Y, LI M L.High-level secretion and characterization of cyclodextrin glycosyltransferase in recombinant Komagataella phaffii[J].Journal of Biotechnology, 2017, 259:126-134.
[34] LIU H, LI J H, DU G C, et al.Enhanced production of α-cyclodextrin glycosyltransferase in Escherichia coli by systematic codon usage optimization[J].Journal of Industrial Microbiology & Biotechnology, 2012, 39(12):1841-1849.
[35] 陈龙军, 陈济琛, 林晓栩, 等.嗜热芽孢杆菌CHB1环糊精酶基因优化及其在毕赤酵母中的表达[J].食品与生物技术学报, 2018, 37(9):994-999.
CHEN L J, CHEN J C, LIN X X, et al.Codon optimization and expression of cyclodextrin glycosyltransferase from Gebacillius sp.CHB1 in Pichia pastoris[J].Journal of Food Science and Biotechnology, 2018, 37(9):994-999.
[36] 郭永华, 陈济琛, 贾宪波, 等.分子伴侣共表达对嗜热环糊精葡萄糖基转移酶异源可溶性表达的影响[J].微生物学通报, 2016, 43(3):518-526.
GUO Y H, CHEN J C, JIA X B, et al.Effects of chaperone co-expression on heterologous solubility expression of thermophilic cyclodextrin glucosetransferase[J].Microbiology China, 2016, 43(3):518-526.
[37] 张艺凡, 周文喜, 王华.环糊精葡萄糖基转移酶的生产及其分离纯化研究进展[J].内蒙古民族大学学报(自然科学版), 2023, 38(3):231-236.
ZHANG Y F, ZHOU W X, WANG H.Research progress in production and separation purification of cyclodextrin glucosyltransferase[J].Journal of Inner Mongolia Minzu University (Natural Sciences), 2023, 38(3):231-236.
[38] 熊海涛, 韦宇拓.枯草芽孢杆菌表达系统及其启动子的研究进展[J].广西科学, 2018, 25(3):233-241.
XIONG H T, WEI Y T.Research progress of Bacillus subtilis expression system and its promoter regulatory elements[J].Guangxi Sciences, 2018, 25(3):233-241.
[39] LI C M, YOU Y X, ZHANG Y Z, et al.Maltose binding site 2 mutations affect product inhibition of Bacillus circulans STB01 cyclodextrin glycosyltransferase[J].International Journal of Biological Macromolecules, 2021, 175:254-261.
[40] KONG D M, WANG L, SU L Q, et al.Effect of Leu277 on disproportionation and hydrolysis activity in Bacillus stearothermophilus NO2 cyclodextrin glucosyltransferase[J].Applied and Environmental Microbiology, 2021, 87(12):e0315120.
[41] 孔德民, 左方圆, 吴敬, 等.Bacillus stearothermophilus NO2环糊精葡萄糖基转移酶Leu277突变提高α-环糊精产量[J].食品与发酵工业, 2023, 49(15):1-7.
KONG D M, ZUO F Y, WU J, et al.Improvement of α-cyclodextrin yield by mutants of Leu277 about CGTase from Bacillus stearothermophilus NO2[J].Food and Fermentation Industries, 2023, 49(15):1-7.
[42] 左方圆. Bacillus stearothermophilus NO2环糊精葡萄糖基转移酶的分子改造及制备α-环糊精的研究[D].无锡:江南大学, 2022.
ZUO F Y.Molecular modification of Bacillus stearothermophilus NO2 cyclodextrin glucosyltransferase and preparation[D].Wuxi:Jiangnan University, 2022.
[43] WANG L, DUAN X G, WU J.Enhancing the α-cyclodextrin specificity of cyclodextrin glycosyltransferase from Paenibacillus macerans by mutagenesis masking subsite-7[J].Applied and Environmental Microbiology, 2016, 82(8):2247-2255.
[44] WANG L, XIA Y M, SU L Q, et al.Modification of Bacillus clarkii γ-cyclodextrin glycosyltransferase and addition of complexing agents to increase γ-cyclodextrin production[J].Journal of Agricultural and Food Chemistry, 2020, 68(43):12079-12085.
[45] LI Y, WEI L K, ZHU Z L, et al.Rational design to change product specificities and thermostability of cyclodextrin glycosyltransferase from Paenibacillus sp[J].RSC Advances, 2017, 7(23):13726-13732.
[46] 王金鹏, 王萍, 苑征, 等.γ-CGTase突变体制备及其产γ-CD条件优化[J].食品与生物技术学报, 2018, 37(10):1015-1020.
WANG J P, WANG P, YUAN Z, et al.Preparation of γ-CGTase mutant and optimization of the production of γ-CD[J].Journal of Food Science and Biotechnology, 2018, 37(10):1015-1020.
[47] CHEN S D, LI Z F, GU Z B, et al.Variants at position 603 of the CGTase from Bacillus circulans STB01 for reducing product inhibition[J].International Journal of Biological Macromolecules, 2019, 136:460-468.
[48] CHEN S D, LI Z F, GU Z B, et al.Leu600 mutations decrease product inhibition of the β-cyclodextrin glycosyltransferase from Bacillus circulans STB01[J].International Journal of Biological Macromolecules, 2018, 115:1194-1201.
[49] LI C M, BAN X F, GU Z B, et al.Calcium ion contribution to thermostability of cyclodextrin glycosyltransferase is closely related to calcium-binding site CaIII[J].Journal of Agricultural and Food Chemistry, 2013, 61(37):8836-8841.
[50] LI Z F, HUANG M, GU Z B, et al.Asp577 mutations enhance the catalytic efficiency of cyclodextrin glycosyltransferase from Bacillus circulans[J].International Journal of Biological Macromolecules, 2016, 83:111-116.
[51] VAN DER VEEN B A, LEEMHUIS H, KRALJ S, et al.Hydrophobic amino acid residues in the acceptor binding site are main determinants for reaction mechanism and specificity of cyclodextrin-glycosyltransferase[J].The Journal of Biological Chemistry, 2001, 276(48):44557-44562.
[52] 李晓涵, 郭姣梅, 宋凯, 等.Bacillus sp.Y112环糊精葡萄糖基转移酶位点R81定点突变提高产物特异性[J].食品科学, 2021, 42(10):133-138.
LI X H, GUO J M, SONG K, et al.Improvement of the product specificity of Bacillus sp.Y112 cyclodextrin glucosyltransferase by site-directed mutagenesis of arginine 81[J].Food Science, 2021, 42(10):133-138.
[53] 郑丹妮. Bacillus sp.FJAT-44876γ-环糊精葡萄糖基转移酶的表征及热稳定性提升研究[D].无锡:江南大学, 2020.
ZHENG D N.Characterization and thermostability improved of γ-CGTase from Bacillus sp. FJAT-44876[D].Wuxi:Jiangnan University, 2020.
[54] SAHNOUN M, JAOUA M, BEJAR S, et al.Highlight on mutations affecting the US132 cyclodextrin glucanotransferase binding specificity, thermal stability, and anti-staling activity[J].Colloids and Surfaces.B, Biointerfaces, 2022, 212:112375.
[55] XIE T, SONG B H, YUE Y, et al.Site-saturation mutagenesis of central tyrosine 195 leading to diverse product specificities of an α-cyclodextrin glycosyltransferase from Paenibacillus sp.602-1[J].Journal of Biotechnology, 2014, 170:10-16.
[56] 柴宝成, 姜钰琳, 倪晔, 等.环糊精葡萄糖基转移酶182位点定点改造催化合成糖基化染料木素[J].生物工程学报, 2022, 38(2):749-759.
CHAI B C, JIANG Y L, NI Y, et al.Engineering the 182 site of cyclodextrin glucosyltransferase for glycosylated genistein synthesis[J].Chinese Journal of Biotechnology, 2022, 38(2):749-759.
[57] HAN R Z, NI J, ZHOU J Y, et al.Engineering of cyclodextrin glycosyltransferase reveals pH-regulated mechanism of enhanced long-chain glycosylated sophoricoside specificity[J].Applied and Environmental Microbiology, 2020, 86(7):e00004-e00020.
[58] 韩瑞枝. 环糊精葡萄糖基转移酶的分子改造及合成糖基化L-抗坏血酸[D].无锡:江南大学, 2013.
HAN R Z.Molecular engineering of cyclodextrin glycosyltransferase for glycosyl L-ascorbic acid synthesis[D].Wuxi:Jiangnan University, 2013.
[59] LI X H, SUN J J, WANG W, et al.Site-saturation mutagenesis of proline 176 in cyclodextrin glucosyltransferase from Bacillus sp.Y112 effects product specificity and enzymatic properties[J].Process Biochemistry, 2020, 94:180-189.
[60] 花敬涵. β-环糊精糖基转移酶的催化机制及理性改造研究[D].合肥:合肥工业大学, 2019.
HUA J H.Catalytic mechanism and rational transformation of β-cyclodextrin glycosyltransferase[D].Hefei:Hefei University of Technology, 2019.
[61] 郑婉, 刘振杨, 郑金珠, 等.易错PCR技术改造β-环糊精葡萄糖基转移酶的催化特性[J].现代食品科技, 2023, 39(5):25-31.
ZHENG W, LIU Z Y, ZHENG J Z, et al.Modification of the catalytic properties of β-cyclodextrin glucosyltransferase through error-prone PCR[J].Modern Food Science and Technology, 2023, 39(5):25-31.
[62] SONG B H, YUE Y, XIE T, et al.Mutation of Tyrosine167 Histidine at remote substrate binding subsite -6 in α-cyclodextrin glycosyltransferase enhancing α-cyclodextrin specificity by directed evolution[J].Molecular Biotechnology, 2014, 56(3):232-239.
[63] 郭永华, 陈济琛, 贾宪波, 等.应用易错PCR技术提高环糊精葡萄糖基转移酶的可溶性表达[J].微生物学报, 2016, 56(10):1551-1560.
GUO Y H, CHEN J C, JIA X B, et al.Improving soluble expression of Geobacillus sp.B1 CGTase by errorprone PCR[J].Acta Microbiologica Sinica, 2016, 56(10):1551-1560.
[64] MELZER S, SONNENDECKER C, FÖLLNER C, et al.Stepwise error-prone PCR and DNA shuffling changed the pH activity range and product specificity of the cyclodextrin glucanotransferase from an alkaliphilic Bacillus sp[J].FEBS Open Bio, 2015, 5(1):528-534.
[65] 陈双娣. 麦芽糖基结合位点2区域对环糊精葡萄糖基转移酶产物抑制的影响研究[D].无锡:江南大学, 2019.
CHEN S D.Effects of maltose binding site 2 region on product inhibition of cyclodextrin glycosyltransferase[D].Wuxi:Jiangnan University, 2019.
[66] 郭永华. 嗜热环糊精葡萄糖基转移酶的分子改造[D].福州:福建农林大学, 2016.
GUO Y H.Molecular modification of the thermophilic cyclodextrin glucosetransferase[D].Fuzhou:Fujian Agriculture and Forestry University, 2016.
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